DCLK1 Inhibition Sensitizes Colorectal Cancer Cells to Radiation Treatment

Colorectal cancer (CRC) is one of the most prevalent diagnosed cancers and a common cause of cancer-related mortality. Despite effective clinical responses, a large proportion of patients undergo resistance to radiation therapy. Therefore, the identification of efficient targeted therapy strategies would be beneficial to overcome cancer radioresistance. Doublecortin-like kinase 1 (DCLK1) is an intestinal and pancreatic stem cell marker that showed overexpression in a variety of cancers. The transfection of DCLK1 siRNA to ‎normal HCT-116 cells was performed, and then cells were irradiated with X-rays. The effects of DCLK1 inhibition on cell survival, apoptosis, cell cycle, DNA damage response (ATM and γH2AX proteins), epithelial-mesenchymal transition (EMT) related genes (vimentin, N‐cadherin, and E-cadherin), cancer stem cells markers (CD44, CD133, ALDH1, and BMI1), and β‐catenin signaling pathway (β‐catenin) were evaluated. DCLK1 siRNA downregulated DCLK1 expression in HCT-116 cells at both mRNA and protein levels (P <0.01). Colony formation assay showed a significantly reduced cell survival in the DCLK1 siRNA transfected group in comparison with the control group following exposure to 4 and 6 Gy doses of irradiation (P <0.01). Moreover, the expression of cancer stem cells markers (P <0.01), EMT related genes (P <0.01), and DNA repair proteins including pATM (P <0.01) and γH2AX (P <0.001) were significantly decreased in the transfected cells in comparison with the nontransfected group after radiation. Finally, the cell apoptosis rate (P <0.01) and the number of cells in the G0/G1 phase in the silencing DCLK1 group was increased (P <0.01). These findings suggest that DCLK1 can be considered a promising therapeutic target for the treatment of radioresistant human CRC.

olorectal cancer (CRC) is considered the third leading cause of cancer-related death for both men and women worldwide (1). Radiotherapy (RT) as a highly effective treatment approach for cancer has been broadly used in the clinic for over 100 years, where directly or indirectly mediating tumor C cell death via inducing multiple types of DNA damage and genome instability (2). Indeed, RT improves the efficacy of surgery by shrinking the tumor before surgery or removing the remaining microscopic tumor cells afterward (3). Despite promising achievement in radiation therapy for controling malignant cells, and curative potentials in several localized cancers, prognosis and the 5-year survival rate remained poor, largely due to intrinsic cellular resistance (4). Therefore, in order to enhance the efficacy of RT, more therapeutic strategies are required for promoting the ionizing radiation (IR) sensitivity of CRC and to overcome IR resistance (5).
A growing body of evidence reveals that various factors including double-strand break (DSB) repair pathway through homologous recombination and nonhomologous end-joining, tumor microenvironment, various deregulated signaling pathways (e.g., AKT or NF-κB,), micro RNA dysregulation, redistribution of the cell cycle, hypoxia, and apoptosis contribute to cellular resistance against radiation (6,7). Besides, cancer stem cells (CSCs) or tumor-initiating cells, and the epithelial-mesenchymal transition (EMT) enable radioresistant tumor cells metastasis after IR exposure (8). Discovering and targeting molecules and signaling pathways associated with these barriers are essential for developing new beneficial radiosensitizers, and improving the efficacy of IR (9).
Doublecortin-like kinase 1 (DCLK1) as a tuft cell marker in the small intestine is a member of the microtubule-associated protein kinase superfamily that regulates embryonic cortical development and neuronal process (10). Cumulative evidence has verified that DCLK1 expression can support CSCs self-renewal, cancer growth, EMT, and metastasis in both early and advanced cancer stages (11).
Moreover, EMT and CSCs that play key roles in the development and progression of cancer, are involved in multimodal therapy resistance and relapse (12).
Here, we hypothesized that IR-induced ataxia telangiectasia mutated (ATM) activation following direct interaction with DCLK1 may lead to the repair of damaged DNA, and increase the survival of cancer cells. Therefore, the present study aimed to explore whether DCLK1 inhibition impacts radiosensitivity, double strand breack (DSB) repair, cell cycle, cell survival, EMT, and CSCs expression in HCT-116 CRC cell line, and elucidate the underlying mechanisms through in vitro investigations.

Cell culture and transfection
The human HCT-116 CRC cell line was Next, the cells were exposed to IR in a single fraction, incubated for seven days, followed by staining with crystal violet. Using Olympus CKX53 inverted microscope (Nagano Olympus Co. Japan), the colonies (>50 cells) were quantified, and to obtain the average colony formation rate the experiment was repeated in triplicate. The   Table 1. Primer sequences for qRT-PCR.

Statistical analysis
Analysis of the data between different treatment groups was conducted using the SPSS16.0 software, and the data were compared by analysis of variance (one-way ANOVA). Alpha was set at 0.05. Values are expressed as mean ± standard error.   Figure 4A). Therefore, it can be concluded that silencing DCLK1 increased radiosensitivity by inducing   Figure 4B).  Conversely, the expression of E-cadherin in the absence of DCLK1 was higher than that in the presence of DCLK1.*P < 0.05 and **P < 0.01 vs. control.

DLCK1 inhibition reduces CSCs and EMT related markers expression
To determine the effect of DCLK1 inhibition on CSCs and EMT-related markers, the expression patterns of key proteins were quantified. The expression of CSCs markers including CD44, prominin (CD133), aldehyde dehydrogenase 1 (ALDH1) and polycomb complex protein (BMI1) was lower in transfected cells in comparison with control cells (P <0.01; Figure 5A). RT-qPCR was performed to determine EMT marker gene expression levels. As presented in Figure   5B, mesenchymal markers such as vimentin, N-cadherin and β-catenin mRNA were down regulated (P < 0.01), while E-cadherin was up regulated (P < 0.05) ( Figure 5A).

Discussion
RT is a highly effective cancer treatment in which about half of all cancer patients receive this treatment modality during their course of illness (3). Despite outstanding achievements in the treatment of CRC by radiotherapeutic procedure, nevertheless, radioresistance remains a major clinical problem (15). To overcome the radioresistance, there is an urgent need to identify the resistance effectors to suppress them and enhance the efficacy of treatment as well as to improve patient outcomes.
DCLK1 is a microtubule-associated protein in which its expression has been reported to be critically required for maintaining the growth of human colon cancer cells (16). In the present study,  (19). Overexpression of DNA damage repairrelated proteins is associated with increased radioresistance in several types of human cancer cells (20,21). It is well known that in the DNA damage repair pathway, ATM as a central kinase plays a major role in controlling genome stability and cell survival (22). ATM silencing was reported to improve the therapeutic efficacy of DNA damaging agents on glioma cells and mantle cell lymphoma (5,23). Indeed, inhibition of ATM kinase in tumor cells by preventing DNA repair, decreasing cell cycle checkpoint activation, and enhancing apoptosis led to radiosensitivity (24). In an early response to DNA damage, phosphorylation of H2AX at Ser-139 by ATM led to produce γH2AX, which creates an epigenetic signal for specific domains on downstream DNA damage repair proteins (25). Lack of H2AX is associated with genomic instability and radiation sensitivity (26). Measurement of γ-H2AX foci levels in cells provides a sensitive marker for detecting DSB repair efficiency because higher γ-H2AX expression associates with more unrepaired DSBs (27). Recently, it has been shown that ATM knockdown sensitized breast cancer cells to irradiation, and reduced phosphorylation of γ-H2AX, highlighting a strong relationship between ATM, DNA repair pathway and radioresistance (28). We observed that DCLK1 down regulation In the present study, we investigated the impact of DCLK1 on EMT phenotype and CSCs markers. Our results revealed for the first time that inhibition of DCLK1 radiosensitized CRC cells partly by modulating EMT genes. The EMT as a highly dynamic process not only regulates normal embryonic development, wound healing and tissue regeneration, but is also involved in all stages of tumorigenesis from initiation to metastatic expansion (35). Furthermore, it has been accepted that loss of epithelial and gain of mesenchymal markers in various cancers was associated with radioresistance (36). In our study, there was a reduction in N-cadherin and vimentin expression, but up regulation of E-cadherin in DCLK1 silenced cells following radiation. Similar results were also reported in the pancreas and breast cancers (37,38).
For example, it has been indicated that sensitivity to RT was more evident in breast cancer cells expressing E-cadherin, relative to the breast cancer cells with no E-cadherin (36). However, less is known about the effect of EMT-related factors on tumor radioresistance.
It has been reported that activation of EMT increased the self-renewal and multi-differentiation potential of CSCs, and there is a close relationship between EMT and stemness factors (39). We found that the expression of CSCs markers decreased in cells transfected with DCLK1 siRNA after radiation. CSCs have unique properties such as high DNA repair capacity, high expression of antiapoptotic genes, and reactive oxygen species (ROS) scavengers which cause cancer radioresistance (2).
Accumulating evidence reveals that Wnt/βcatenin signaling as a major regulator of EMT and CSCs process may be a possible target to overcome the resistance to RT (41). β-catenin has a critical role in protecting cells from radiation-induced death through elevating DSBs repair, ROS scavenging, and suppressing apoptosis (41). In addition, the deregulation of Wnt signaling is linked with the radioresistance in numerous cancers (41,42). It has been proven that DCLK1 protein is an essential effector in maintaining β-catenin expression for cell survival (11,29). Similarly, we revealed that the down regulation of DCLK1 in CRC cells reduced β-catenin expression after IR.
In summary, we found for the first time that the silencing of DCLK1 enhanced the radiosensitivity of CRC cells. Decreased DSB repair, EMT and CSCs related genes down regulation, enhanced G0/G1 arrest and apoptosis, and suppressed β-catenin signaling, contribute to an increase in radiosensitivity induced by DCLK1 inhibition. Therefore, the present observation suggests that the combination of DCLK1 down regulation with ionizing radiation could serve as a promising therapeutic strategy to reverse radioresistance in CRC. Further radiobiological studies are required to highlight the role of DCLK1 and its downstream signaling pathway with radiosensitivity of other tumor cell lines.